Blood collection device comprising an inhibitor of hexokinase, a glycolysis-inhibiting agent, and an anticoagulant or plasma stabilizer

ABSTRACT

The present invention relates to a composition for the stabilization of glucose, lactate and homocysteine in blood after collection, to a use of the provided compositions and a method for the stabilization of glucose, lactate and homocysteine in blood after collection, as well as optionally the in vitro determination of glucose, lactate and homocysteine in blood, and a blood collection device provided for said use and method.

This Application is a Division of U.S. patent application Ser. No.14/420,528, filed on Feb. 9, 2015, which application is the nationalphase under 35 U.S.C. § 371 of PCT International Application No.PCT/EP2013/065983, which has an International filing date of Jul. 30,2013, which claims priority to European Patent Application No.12179907.6, filed Aug. 9, 2012. The entire contents of theseapplications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the ex vivo stabilization and storageof blood. In particular, the present invention relates to a compositionfor the stabilization of glucose, lactate and homocysteine in bloodafter collection, to a use of the provided compositions and a method forthe stabilization of glucose, lactate and homocysteine in blood aftercollection, as well as optionally the in vitro determination of glucose,lactate and homocysteine in blood, and a blood collection deviceprovided for said use and method.

BACKGROUND OF THE INVENTION

Blood sampling and testing are routinely carried out for variousdiagnostic purposes. Detection and quantification of glucose in blood isfor example used in the diagnosis and management of disorders ofcarbohydrate metabolism such as diabetes mellitus. Determination oflactate in blood may for example be used to test acid-base homeostasisand to screen for lactic acidosis, hypoxia or sepsis, and to evaluateadaptation to exercise. An increased level of plasma total homocysteine(tHcy) is for example a risk factor for cardiovascular disease and asensitive marker for vitamin B deficiencies. In order to be usefuldiagnostic indicators and predictors, the levels of such bloodcomponents should be determined accurately and precisely.

Since metabolism in blood cells (erythrocytes, leukocytes and platelets)is ongoing ex vivo, i.e. after blood collection, levels of bloodglucose, lactate and homocysteine can change significantly during thetime elapsed between withdrawal and analysis, potentially leading toerroneous results, especially when the time elapsed is variable anduncontrolled and when storage conditions such as temperature vary. Dueto continued glycolysis in blood cells, mainly in the erythrocytes, theconcentration of blood glucose decreases after collection. Chan et al.in Clinical Chemistry, 38, 1992, pp. 411-413 reported that the plasmaglucose concentration in heparinized blood samples at room temperaturedecreases at a rate of approximately 0.3 mmol/L per hour during thefirst 12 hours after blood collection. The decrease in blood glucose isaccompanied by an increase in lactate concentration. Astles et al. inClinical Chemistry, 40, 1994, pp. 1327-1330 indicated the increase inlactate to be 0.7 mmol/L per hour, which is large in view of the citedreference interval of 0.5-2.2 mmol/L for lactate. Because of ongoinghomocysteine metabolism, wherein homocysteine is formed from methioninevia adenosylmethionine-dependent methyl transfer reactions, and ongoingrelease of homocysteine from erythrocytes, tHcy increases after bloodcollection. Nauck et al. in Clinical Chemistry and Laboratory Medicine,39, 2001, pp. 675-680 reported that at room temperature theconcentration of tHcy increases by approximately 1-2 μmol/L per hourduring the first hours, which corresponds to an approximately 10%increase of tHcy per hour. This means typically after blood collectionglucose is continuously consumed by the blood cells and lactate andhomocysteine are released from the blood cells.

The changes in glucose, lactate and homocysteine levels in blood aftercollection are time- and temperature-dependent. Plasma concentrationchanges of these substances can be prevented by the immediatecentrifugation and removal of the blood cells. This requires theaddition of anticoagulant, such as EDTA salt, citrate salt, oxalate saltand heparin salt, to the whole blood. Moreover, a centrifuge and thecapacity for immediate processing need to be available at or close tothe site of blood collection which can be problematic and impractical.Blood samples can be cooled on ice until centrifugation to reducechanges in glucose, lactate and homocysteine levels. However, chillingon ice may not be sufficient and may be impossible or impractical, forexample when there is a substantial delay between the collection and theprocessing and analysis, or when many samples have to be collected andtransported. For blood serum, changes in glucose, lactate andhomocysteine levels can occur because serum is prepared from a bloodsample left at room temperature for a time sufficient to allowcoagulation (c. 20-60 min), and centrifugation and separation of theblood cells is possible only thereafter. Preanalytical sample processingcan interfere in the subsequent testing for glucose, lactate andhomocysteine or co-analytes. For example, disturbance of cellularintegrity such as hemolysis may be problematic. Furthermore,time-consuming or error-prone processing steps, for exampledeproteination using trichloroacetic acid, may not be suitable inroutine clinical processing.

When a blood sample cannot be separated or cooled immediately aftercollection, in addition to an anticoagulant an antiglycolytic agent suchas iodoacetate, mannose or fluoride as stabilizer can be added. However,mannose can interfere with enzymatic analysis methods using glucoseoxidase or hexokinase. Fluoride stabilizes glucose primarily byinhibiting enolase in the glycolytic pathway. Chan et al. in ClinicalChemistry, 35, 1989, pp. 315-317 showed that fluoride does not preventloss of plasma glucose completely and that at room temperature theconcentration of glucose in blood containing fluoride decreasessignificantly for the first four hours after collection. Westgard et al.in Clinical Chemistry, 18, 1972, pp. 1334-1338 described that wholeblood stabilized with sodium fluoride and separated within 15 minprovides an acceptable sample for lactate determination. However, forthe case without separation Astles et al. reported that for bloodsamples with sodium fluoride and potassium oxalate at room temperaturelactate increased by 0.2 mmol/L after 1 hour post-collection. Fluoride,adenosine analogues such as 3-deazaadenosine or citrate are used tostabilize tHcy after blood collection, but the use of stabilizersusually leads to deviations at baseline and the problem withstabilization of tHcy is only partly solved. Some typically usedstabilizers of tHcy can affect analytical techniques such asfluorescence polarization immunoassay, chemiluminescence immunoassay andenzyme linked immunoassay (see e.g. Nauck et al.).

The use of two agents for the inhibition of glycolysis in blood sampleshas been considered. Chan et al. (1992) reported the use of sodiumfluoride and mannose, but stabilization of the blood glucoseconcentration was incomplete. Moreover, mannose can interfere withenzymatic analysis methods using glucose oxidase or hexokinase. Buedingand Goldfarb in Journal of Biological Chemistry, 141, 1941, pp. 539-544demonstrated the use of sodium iodoacetate and sodium fluoride topreserve glucose and lactate. The use of glyceraldehyde and sodiumfluoride for the preservation of plasma glucose concentrations wasreported by le Roux et al. in Annals of Clinical Biochemistry, 41, 2004,pp. 43-46. Glucose stabilization using sodium fluoride and acidificationwith citric acid was described by Gambino et al. in Clinical Chemistry,55, 2009, pp. 1019-1021. However, while these studies aimed atstabilizing glucose and/or lactate, in vitro stabilization of glucose,lactate and homocysteine in blood has not been addressed.

There is a need in the art to make more robust and to simplifypreanalytics for the accurate testing of blood glucose, lactate andhomocysteine while avoiding cumbersome, complicated, error-prone,costly, impractical or time-critical near-collection processing such asimmediate centrifugation or icing of blood samples and providingbenefits in terms of improved handling, storage and transport of saidsamples as well as sample throughput. In particular, the object of thepresent invention is to effect sufficient and predictable inhibition ofglycolysis and to efficiently stabilize blood glucose, lactate andhomocysteine in blood after collection at room temperature enablingprolonged storage of blood and allowing blood glucose, lactate andhomocysteine and other substances to be determined accurately andreliably using a single sample.

SUMMARY OF THE INVENTION

The object is solved by the in vitro methods according to claims 1 and12, the uses according to claims 13 and 20, the blood collection deviceaccording to claim 16, the kit according to claim 17 and the compositionaccording to claim 18, while preferred embodiments are provided in thedependent claims and are further described below.

The present invention in particular provides the following itemsincluding main aspects and preferred embodiments, which respectivelyalone and in combination particularly contribute to solving the aboveobject and eventually provide additional advantages:

-   (1) A method for the treatment of a blood sample in vitro, wherein    blood is mixed in vitro with a composition comprising    -   i) at least one inhibitor of hexokinase;    -   ii) at least one glycolysis-inhibiting agent having activity for        another enzyme involved in glucose catabolism; and    -   optionally iii) an anticoagulant and/or a plasma stabilizer, and    -   wherein said blood sample is preserved for further analysis.-   (2) The method according to item (1), wherein one and/or more of    glucose, lactate and homocysteine in the blood sample is (are)    stabilized, preferably glucose is stabilized.-   (3) The method according to item (1) or (2), wherein the composition    further comprises iv) ammonium salt NR₄X,    -   wherein each R independently is hydrogen, linear C₁-C₆ alkyl,        branched C₃-C₆ alkyl, unsubstituted phenyl or substituted        phenyl, and X is halide, hydroxide, C₁-C₄ alkoxide and acetate,        and wherein preferably each R independently is hydrogen, methyl        or ethyl, and X is fluoride or chloride, and wherein more        preferably NR₄X is tetramethylammonium fluoride,        tetramethylammonium chloride, tetraethylammonium chloride or        NH₄Cl.-   (4) The method according to item (3), wherein the concentration of    the ammonium salt NR₄X is 0.01 to 100 μmol/mL blood, preferably is    0.1 to 10 μmol/mL blood, and more preferably is 0.5 to 1 μmol/mL    blood.-   (5) The method according to any one of the preceding items, wherein    the at least one inhibitor of hexokinase i) is selected from the    group consisting of 2-deoxy-D-glucose, 2-fluoro-2-deoxy-D-glucose,    2-amino-2-deoxy-D-glucose and 3-bromopyruvic acid or salt thereof.-   (6) The method according to any one of the preceding items, wherein    the at least one glycolysis-inhibiting agent ii) has antiglycolytic    activity for any one of the enzymes in the glycolytic pathway    downstream of hexokinase.-   (7) The method according to any one of the preceding items, wherein    the at least one glycolysis-inhibiting agent ii) is selected from    the group consisting of fluoride salt, iodoacetic acid or salt    thereof, oxamic acid or salt thereof and dichloroacetic acid or salt    therof.-   (8) The method according to any one of the preceding items, wherein    the anticoagulant iii) is selected from the group consisting of EDTA    salt, citrate salt, oxalate salt and heparin salt.-   (9) The method according to any one of items (5), (7) or (8),    wherein salt as set forth in any one of items (5), (7) or (8) is    salt of NR₄ ⁺, wherein each R independently is hydrogen, linear    C₁-C₆ alkyl, branched C₃-C₆ alkyl, unsubstituted phenyl or    substituted phenyl, and wherein preferably each R independently is    hydrogen, methyl or ethyl, and wherein more preferably NR₄ ⁺ is    tetramethylammonium ion, tetraethylammonium ion or NH₄ ⁺, and    wherein most preferably NR₄ ⁺ is NH₄ ⁺.-   (10) The method according to any one of the preceding items, wherein    the mass concentration of the at least one inhibitor of    hexokinase i) is at least 0.01 mg/mL blood and wherein the mass    concentration of the at least one glycolysis-inhibiting agent ii) is    at least 0.01 mg/mL blood.-   (11) The method according to any one of the preceding items, wherein    the mass concentration of the at least one inhibitor of    hexokinase i) is at least 0.01 mg/mL blood, more preferably is at    least 0.02 mg/mL blood, even more preferably is in a range from 0.02    to 25 mg/mL blood and most preferably is in a range from 0.02 to 4    mg/mL blood,    -   wherein the mass concentration of the at least one        glycolysis-inhibiting agent ii) is at least 0.01 mg/mL blood,        more preferably is at least 0.02 mg/mL blood, even more        preferably is in a range from 0.02 to 25 mg/mL blood and most        preferably is in a range from 0.02 to 4 mg/mL blood,    -   wherein optionally the mass concentration of the optional        anticoagulant iii) is at least 0.01 mg/mL blood, more preferably        is at least 0.1 mg/mL blood, even more preferably is in a range        from 0.1 to 25 mg/mL blood and most preferably is in a range        from 1 to 2.5 mg/mL blood with or 12 to 30 IU/mL blood,    -   and wherein preferably the ratio of the mass concentrations of        the at least one inhibitor of hexokinase i) and the at least one        glycolysis-inhibiting agent ii) is in a range from 100:1 to        1:100, more preferably is in a range from 50:1 to 1:50, even        more preferably is in a range from 25:1 to 1:25 and most        preferably is in a range from 10:1 to 1:10.-   (12) The method according to any one of the preceding items, wherein    the composition is in solid form, lyophilized form or in solution,    and wherein in a case of a solution water or dilute acidic aqueous    solution is preferred.-   (13) The method according to any one of the preceding items, wherein    the composition is substantially free of agents for the lysis of    blood cells.-   (14) The method according to any one of the preceding items, further    containing a buffer and/or an additive commonly used for preserving    blood, preferably in a content of up to 50% by weight.-   (15) A method for the inhibition of glycolysis and optionally    coagulation in a blood sample in vitro, wherein blood is mixed after    its withdrawal with the composition as set forth in any one of    items (1) to (14).-   (16) An in vitro diagnostic method, wherein the amount(s) of blood    component(s) is (are) determined in a stabilized blood sample mixed    with the composition as set forth in any one of items (1) to (14).-   (17) The method according to item (15) or (16), wherein one and/or    more of glucose, lactate and homocysteine are stabilized.-   (18) The method according to any one of items (15) to (17), wherein    one and/or more of glucose, lactate and homocysteine are stabilized    at room temperature for up to 50 hours after blood collection.-   (19) The method according to any one of items (15) to (18), wherein    glucose, lactate and homocysteine are stabilized simultaneously.-   (20) The method according to any one of the preceding items, wherein    the amount of one and/or more of glucose, lactate and homocysteine    in blood and/or other blood component(s) are determined by    comprising the following steps:    -   (a) providing a blood collection device comprising, placed in        the device, the composition as set forth in any one of items (1)        to (14);    -   (b) placing blood in the blood collection device;    -   (c) mixing the composition as set forth in any one of items (1)        to (14) with blood in the blood collection device;    -   optionally (d) storing the blood in the blood collection device        for a predetermined period of time during which glucose, lactate        and homocysteine levels are substantially constant; and    -   (e) determining the amount of one and/or more of glucose,        lactate and homocysteine and/or other blood component(s) in the        blood sample.-   (21) The method according to item (20), wherein, instead of    steps (a) and (b), a step is carried out which comprises placing    blood in a blood collection device and subsequently placing a    composition of the present invention in the blood collection device.-   (22) The method according to item (20) or (21), wherein the mass    concentration of the at least one inhibitor of hexokinase i) is at    least 0.01 mg/mL blood, more preferably is at least 0.02 mg/mL    blood, even more preferably is in a range from 0.02 to 25 mg/mL    blood and most preferably is in a range from 0.02 to 4 mg/mL blood,    -   wherein the mass concentration of the at least one        glycolysis-inhibiting agent ii) is at least 0.01 mg/mL blood,        more preferably is at least 0.02 mg/mL blood, even more        preferably is in a range from 0.02 to 25 mg/mL blood and most        preferably is in a range from 0.02 to 4 mg/mL blood,    -   wherein optionally the mass concentration of the optional        anticoagulant iii) is at least 0.01 mg/mL blood, more preferably        is at least 0.1 mg/mL blood, even more preferably is in a range        from 0.1 to 25 mg/mL blood and most preferably is in a range        from 1 to 2.5 mg/mL blood or 12 to 30 IU/mL blood,    -   and wherein preferably the ratio of the mass concentrations of        the at least one inhibitor of hexokinase i) and the at least one        glycolysis-inhibiting agent ii) is in a range from 100:1 to        1:100, more preferably is in a range from 50:1 to 1:50, even        more preferably is in a range from 25:1 to 1:25 and most        preferably is in a range from 10:1 to 1:10.-   (23) The method according to any one of items (20) to (22), wherein    step (d) is carried out at room temperature for up to 50 hours.-   (24) The method according to any one of items (20) to (23), wherein    the blood in step (b) is whole blood.-   (25) The method according to any one of items (20) to (24), wherein    glucose, lactate and homocysteine in step (e) are plasma glucose,    plasma lactate and plasma homocysteine respectively.-   (26) The method according to any one of items (20) to (25), wherein    in step (e) the determination of the amount(s) of the respective    blood component(s) is carried out using conventional physical,    chemical, enzymatic and/or immunological methods, including    combinations thereof.-   (27) The method according to any one of the preceding items, wherein    lysis of blood cells is substantially inhibited, and preferably    lysis of blood cells is inhibited.-   (28) A use of the composition as set forth in any one of items (1)    to (14) for the inhibition of glycolysis and optionally coagulation    in a blood sample in vitro.-   (29) The use according to item (28), wherein any one of glucose,    lactate and homocysteine is stabilized in blood.-   (30) The use according to item (28), wherein any combination of    glucose, lactate and homocysteine is stabilized.-   (31) The use according to item (28), wherein glucose, lactate and    homocysteine are stabilized simultaneously.-   (32) The use according to any one of item (28) to (31), wherein    glucose, lactate and/or homocysteine are stabilized at room    temperature for up to 50 hours after blood collection.-   (33) The use according to any one of item (28) to (32), wherein,    concurrently with or subsequently to the use of a composition as set    forth in any one of items (1) to (14), (a) test(s) is (are) carried    out for the determination of one and/or more of glucose, lactate and    homocysteine and optionally a further blood component.-   (34) A blood collection device comprising the composition as set    forth in any one of items (1) to (14).-   (35) The blood collection device according to item (34), comprising    a device which is capable of being connected with a conventional    blood withdrawal device.-   (36) A use of the blood collection device according to item (34)    or (35) for stabilization and/or storage of a blood sample in vitro.-   (37) The use according to item (36), wherein the blood sample is    stabilized and/or stored at room temperature for up to 50 hours    after blood collection.-   (38) The use according to item (36) or (37), wherein, concurrently    with or subsequently to the use of a composition as set forth in any    one of items (1) to (14), (a) test(s) is (are) carried out for the    determination of one and/or more of glucose, lactate and    homocysteine and optionally a further blood component.-   (39) A kit comprising:    -   the blood collection device according to item (34) or (35), and    -   test substances for the determination of at least one of,        optionally all simultaneously, glucose, lactate and homocysteine        and optionally a further blood component in collected blood.-   (40) A composition as set forth in any one of items (1) to (14) for    the stabilization of one and/or more of glucose, lactate and    homocysteine in blood.-   (41) The composition according to item (40), wherein the at least    one glycolysis-inhibiting agent ii) is NH₄F.-   (42) A use of at least one glycolysis-inhibiting agent for the    stabilization and ex vivo determination of homocysteine in blood    after withdrawal.-   (43) The use according to item (42), wherein the at least one    glycolysis-inhibiting agent has activity for an enzyme involved in    glucose catabolism.-   (44) The use according to item (42), wherein the at least one    glycolysis-inhibiting agent has antiglycolytic activity for any one    of the enzymes in the glycolytic pathway.-   (45) The use according to item (44), wherein the at least one    glycolysis-inhibiting agent is at least one inhibitor of hexokinase.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the present invention is described in more detailwhile referring to preferred embodiments and examples, which arepresented however for illustrative purposes and shall not be construedto limit the invention in any way.

A first aspect of the present invention is a method for the treatment ofa blood sample in vitro, wherein blood is mixed in vitro with acomposition comprising at least one inhibitor of hexokinase, at leastone glycolysis-inhibiting agent having activity for another enzymeinvolved in glucose catabolism and optionally an anticoagulant and/or aplasma stabilizer, and wherein said blood sample is efficientlypreserved for further analysis.

According to the invention, glycolysis and optionally coagulation areefficiently inhibited in a blood sample in vitro when blood is mixedafter its withdrawal with the composition according to the presentinvention. In particular, one and/or more of glucose, lactate andhomocysteine in the blood sample are thus stabilized.

The composition according to the invention is provided for thestabilization of one and/or more of, preferably all of, glucose, lactateand homocysteine in blood, wherein the composition comprises at leastone inhibitor of hexokinase, at least one glycolysis-inhibiting agenthaving activity for another enzyme involved in glucose catabolism andoptionally an anticoagulant and/or a plasma stabilizer. Stabilization inblood in vitro can thus be achieved.

Hexokinase catalyzes the first step in glycolysis, i.e. the metabolicpathway that converts glucose to pyruvate, said first step being thephosphorylation of glucose. Glucose and glucose 6-phosphate can becatabolized, i.e. broken down to provide energy but also to serve as asource of carbon, and they are also involved in several metabolicpathways other than glycolysis. Surprisingly, it was found in thepresent invention that a composition comprising a combination of atleast one inhibitor of hexokinase and at least one agent having activityfor another enzyme involved in glucose catabolism with an inhibitoryeffect on glycolysis stabilizes one and/or more of glucose, lactate andhomocysteine in blood after collection particularly efficiently, fastand continuously.

This is unexpected considering in particular that substances known andused in the art such as fluoride, iodoacetate or mannose have beendemonstrated to be insufficient by resulting in a decrease in bloodglucose concentration with time. Even compositions with two agents forthe inhibition of glycolysis known in the art, while potentially havingimproved antiglycolytic action compared to cases where only oneantiglycolytic agent is used, may on the one hand still not be efficientin the early time intervals after blood collection (iodoacetate andfluoride) or on the other hand be unspecific and thus not wellcontrolled or predictable in their inhibition (glyceraldehyde andfluoride; fluoride and acidification). Furthermore, prior to theparticular combination of the present invention an efficientstabilization has been not only affected by insufficient inhibition ofglycolysis, but factors such as loss of cellular integrity, change inosmolarity and ion leakage as well as interference in subsequent analytetesting also need to be taken into account. For example, excessiveacidification and use of glyceraldehyde can interfere in thedetermination of lactate.

According to a particularly advantageous embodiment blood is mixed invitro with a composition that comprises not only the at least oneinhibitor of hexokinase, the at least one glycolysis-inhibiting agenthaving activity for another enzyme involved in glucose catabolism andoptionally the anticoagulant and/or the plasma stabilizer, butfurthermore an ammonium salt NR₄X, wherein each R independently ishydrogen, linear C₁-C₆ alkyl, branched C₃-C₆ alkyl, unsubstituted phenylor substituted phenyl, and X is halide, hydroxide, C₁-C₄ alkoxide andacetate. Preferably each R independently is hydrogen, methyl or ethyl,and X is fluoride or chloride. More preferably NR₄X istetramethylammonium fluoride, tetramethylammonium chloride,tetraethylammonium chloride or NH₄Cl. The concentration of the ammoniumsalt NR₄X is not particularly limited. In particular, the upper limit isnot especially limited. A lower preferred limit is 0.01 μmol/mL blood.In a preferred embodiment the concentration of the ammonium salt NR₄X is0.01 to 100 μmol/mL blood, more preferably is 0.1 to 10 μmol/mL blood,and even more preferably is 0.5 to 1 μmol/mL blood.

According to the present invention, preferably lysis of blood cells issubstantially avoided, and more preferably lysis of blood cells isavoided. Preferably, lysis of blood cells is substantially inhibited,and more preferably lysis of blood cells is inhibited. The compositionof the invention therefore is preferably substantially free of agentsfor the lysis of blood cells, and more preferably is free of agents forthe lysis of blood cells.

Surprisingly, it was found that when the ammonium salt iv) of theinvention is additionally comprised in the composition hemolysis can beefficiently and effectively inhibited (see also Examples 10-13 and Table10). This advantageous effect is especially relevant in cases whereoccurrence of hemolysis must be significantly reduced or even safely andsurely avoided, or where presence of agents which may affect cellularstability cannot be entirely ruled out or avoided. Hemolysis cansignificantly further destabilize the sample and prevent prolongedstorage. Furthermore, hemolysis can be detrimental to blood testing anddiagnostics because, for example, mixing of plasma components withcellular components from the lysed cells can lead to spurious resultsfor both plasma and cell analyses or even prevent such analysesaltogether. Therefore, inhibiting hemolysis is advantageous for thereliable determination of blood components.

Therefore, the provision of the ammonium salt iv) as a further additiveprovides significant further benefits. In particular, one and/or more ofglucose, lactate and homocysteine, preferably all, in the blood sampleis (are) stabilized, while furthermore the inhibition of hemolysis canbe significantly enhanced. This further improved stabilization andpreservation of a blood sample can provide longer storage and a morereliable determination of blood components and thus improveddiagnostics.

In an embodiment the at least one inhibitor of hexokinase is selectedfrom the group consisting of 2-deoxy-D-glucose,2-fluoro-2-deoxy-D-glucose, 2-amino-2-deoxy-D-glucose and 3-bromopyruvicacid or salt thereof. 2-deoxy-D-glucose, 2-fluoro-2-deoxy-D-glucose and2-amino-2-deoxy-D-glucose are glucose analogues and hexokinaseinhibitors that can be phosphorylated but not metabolized.3-bromopyruvic acid is a strong alkylating agent which inhibitshexokinase.

In one embodiment the at least one glycolysis-inhibiting agent hasantiglycolytic activity for any one of the enzymes in the glycolyticpathway downstream of hexokinase, comprising phosphoglucose isomerase,phosphofructokinase, aldolase, triose phosphate isomerase,glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase,phosphoglycerate mutase, enolase and pyruvate kinase. In another,optionally combined embodiment the at least one glycolysis-inhibitingagent is selected from the group consisting of fluoride salt, iodoaceticacid or salt therof, oxamic acid or salt thereof and dichloroacetic acidor salt therof. Fluoride has an inhibitory effect on enolase,iodoacetate inhibits glyceraldehyde 3-phosphate dehydrogenase, whileoxamate inhibits lactate dehydrogenase which catalyzes the conversion ofpyruvate to lactate. Dichloroacetate stimulates the activity of pyruvatedehydrogenase by inhibiting pyruvate dehydrogenase kinase thusdecreasing lactate formation.

The inhibitor of hexokinase preferably is a reversible or irreversibleinhibitor of hexokinase, wherein hexokinase comprises the isoforms orisozymes of said enzyme, including glucokinase. In the case ofreversible inhibition, inhibition may be competitive, uncompetitive,mixed and non-competitive.

Preferably, inhibitor of hexokinase is specific, i.e. inhibition isdifferent from irreversible enzyme inactivation by non-specificeffect(s) such as generally destroying protein structure ordenaturation, for example caused by changes of pH or temperature. Theinhibitory effect of an inhibitor of hexokinase can be tested byproviding said inhibitor, a hexose, preferably glucose, as a substrate,ATP and hexokinase in vitro, preferably in physiological ornear-physiological conditions, and measuring the time-dependentconcentration of the hexose and phosphorylated hexose, wherein aninhibitory effect is present when the hexose concentration decreaseswith time more slowly or not at all and/or the concentration ofphosphorylated hexose increases with time more slowly or not at allcompared to a system lacking the inhibitor.

The glycolysis-inhibiting agent having activity for another enzymeinvolved in glucose catabolism preferably is a specific enzyme inhibitorand/or enzyme activator for any one of the enzymes involved in glucosecatabolism, wherein said agent inhibits glycolysis. In the case of anenzyme inhibitor, inhibition can be irreversible or reversible, whereinreversible inhibition may be competitive, uncompetitive, mixed andnon-competitive. Preferably, the enzyme inhibitor is specific, i.e.inhibition is different from irreversible enzyme inactivation bynon-specific effect(s) such as generally destroying protein structure ordenaturation, for example caused by changes of pH or temperature. In thecase of an enzyme activator, said activator preferably is an agent thatinteracts specifically with an enzyme and increases the activity of thelatter, for example through an allosteric effect. The inhibitory effectof the glycolysis-inhibiting agent on glycolysis can be tested bymonitoring over time glucose and/or pyruvate or respectively lactateconcentrations.

The optional anticoagulant is preferably present and is typicallyfurther to/other than the at least one inhibitor of hexokinase and theat least one glycolysis-inhibiting agent, more preferably is selectedfrom the group consisting of EDTA salt, citrate salt, oxalate salt andheparin salt. In one embodiment, lithium heparinate or ammoniumheparinate and EDTA salt are more preferred, and in particular lithiumheparinate or ammonium heparinate is most preferred, because it isamenable to the determination of a multitude of co-analytes, while forexample EDTA salt may limit the number of determinable analytes.Unexpectedly, it was found in the present invention that when thecomposition of the invention comprises heparinate, then glucose, lactateand homocysteine are stabilized particularly fast, efficiently andcontinuously.

In the case that one and/or more of the at least one inhibitor ofhexokinase, the at least one glycolysis-inhibiting agent having activityfor another enzyme involved in glucose catabolism and optionally theanticoagulant and/or the plasma stabilizer is (are) provided as salt, inparticular salts as set forth in items (5), (7) and (8), hemolysis canunexpectedly and advantageously be significantly inhibited by providingat least one salt of said agents as an ammonium salt (see also Examples8 and 9, and Table 10). Ammonium salt denotes a salt of NR₄ ⁺, whereineach R independently is hydrogen, linear C₁-C₆ alkyl, branched C₃-C₆alkyl, unsubstituted phenyl or substituted phenyl, and whereinpreferably each R independently is hydrogen, methyl or ethyl, andwherein more preferably NR₄ ⁺ is tetramethylammonium ion,tetraethylammonium ion or NH₄ ⁺, and wherein most preferably NR₄ ⁺ isNH₄ ⁺. It is particularly preferred that all salts as set forth in items(5), (7) and (8) are salts of NR₄ ⁺ as specified.

Therefore, the provision of ammonium salt for one and/or more of theagents as set forth in items (5), (7) and (8) can provide significantfurther benefits in terms of stabilization and preservation of a bloodsample and of facilitating the reliable determination of bloodcomponents.

It is particularly advantageous when ammonium salt for one and/or moreof the agents as set forth in items (5), (7) and (8) and additionallythe ammonium salt iv) as a further additive are provided together in thecomposition. This way, for example, changes in osmolarity and theoutflow of water from the blood cells which would lead to a possibledilution effect and possible impairment of cellular integrity due to thedesiccation of blood cells can be even more minimized or avoided. Thiscan lead to an enhanced inhibition of hemolysis and even furtherimproved stabilization of the blood sample.

The composition according to the present invention can comprise a bufferand/or an additive commonly used in the preanalytics of blood and forpreserving blood, preferably in a content of up to 50% by weight.Preferably the composition is stable at room temperature and retains itseffects for several weeks, more preferably for several months, and mostpreferably for several years, thus extending shelf life.

In a specific embodiment one and/or more of glucose, lactate andhomocysteine are stabilized at room temperature for up to 50 hours afterblood collection. However, a temperature from 0° C. to 37° C. and apost-collection time of up to 96 hours are comprised in the methodaccording to the present invention. Unexpectedly and advantageously themethod of the present invention stabilizes glucose, lactate andhomocysteine simultaneously. In an embodiment wherein the amount of oneand/or more of glucose, lactate and homocysteine in blood and/or otherblood component(s) are determined the following steps are comprised.

A blood collection device comprising, placed in the device, acomposition according to the present invention is provided, followed byplacing blood in said blood collection device. Alternatively, blood isplaced in a blood collection device and subsequently a composition ofthe present invention is placed in the blood collection device. Thecomposition of the present invention is mixed with blood in the bloodcollection device. Optionally the blood is stored in the bloodcollection device for a predetermined period of time, for example untilprocessing such as centrifugation or analysis and for up to 50, 72 or 96hours at room temperature, during which glucose, lactate andhomocysteine levels are substantially constant. In a further step theamount of one and/or more of glucose, lactate and homocysteine and/orother blood component(s) in the blood sample is determined, for exampleby using conventional physical, chemical, enzymatic and/or immunologicalmethods, including combinations thereof. Without being limited thereto,analytical methods may comprise gas chromatography (GC), massspectrometry (MS), gas chromatography-mass spectrometry (GC-MS), gaschromatography-mass spectrometry with isotopic dilution (GC-ID-MS),liquid chromatography (LC), liquid chromatography with mass spectrometry(LC-MS), liquid chromatography with tandem mass spectrometry (LC-MS-MS),high-pressure liquid chromatography (HPLC), high-pressure liquidchromatography with fluorescence detection (HPLC-FD), HPLC withelectrochemical detection (HPLC-ED), fluorescence polarizationimmunoassay (FPIA), chemiluminescence immunoassay (e.g. ICL), enzymelinked immunoassay (EIA), ion-exchange chromatography (IEC), capillaryelectrophoresis and capillary electrophoresis with laser-inducedfluorescence, wherein HPLC and immunoassays are particularly widely usedin clinical laboratories.

After the mixing of the composition of the present invention and theblood, the mass concentration of the at least one inhibitor ofhexokinase preferably is at least 0.01 mg/mL blood, more preferably isat least 0.02 mg/mL blood, even more preferably is in a range from 0.02to 25 mg/mL blood and most preferably is in a range from 0.02 to 4 mg/mLblood, the mass concentration of the at least one glycolysis-inhibitingagent preferably is at least 0.01 mg/mL blood, more preferably is atleast 0.02 mg/mL blood, even more preferably is in a range from 0.02 to25 mg/mL blood and most preferably is in a range from 0.02 to 4 mg/mLblood, optionally the mass concentration of the optional anticoagulantpreferably is at least 0.01 mg/mL blood, more preferably is at least 0.1mg/mL blood, even more preferably is in a range from 0.1 to 25 mg/mLblood and most preferably is in a range from 1 to 2.5 mg/mL blood or 12to 30 IU/mL blood, and preferably the ratio of the mass concentrationsof the at least one inhibitor of hexokinase and the at least oneglycolysis-inhibiting agent is in a range from 100:1 to 1:100, morepreferably is in a range from 50:1 to 1:50, even more preferably is in arange from 25:1 to 1:25 and most preferably is in a range from 10:1 to1:10. Preferably, whole blood is mixed, and preferably plasma glucose,plasma lactate and plasma homocysteine respectively are determined.Using whole blood and plasma analysis instead of serum is advantageousin that there is no time required to wait for coagulation, there is moresample material and there are no artifacts stemming from coagulationeffects such as slight hemolysis or ongoing coagulation aftercentrifugation. Substantially constant levels preferably are levelswhich have a time-dependent variation of not more than 4% for glucoseand homocysteine and not more than 8% for lactate, more preferably arelevels which have a time-dependent variation of not more than 3% forglucose and homocysteine and not more than 5% for lactate, even morepreferably are levels which have a time-dependent variation of not morethan 2% for glucose and homocysteine and not more than 4% for lactate,yet even more preferably are levels which have a time-dependentvariation of not more than 1.5% for glucose and homocysteine and notmore than 3% for lactate and most preferably are levels which have atime-dependent variation of not more than 1% for glucose andhomocysteine and not more than 2% for lactate.

The composition according to the present invention is preferablyprovided in solid form, lyophilized form or in solution, and wherein ina case of a solution water or dilute acidic aqueous solution ispreferred. In an embodiment dilute acidic aqueous solution is used toenhance the solubility of substances in the composition of theinvention, i.e. it is a solvent or a solubilizer. However, in a case ofa solution, water is more preferred, because excessive acidification caninterfere in the determination of lactate. In an embodiment, thecomposition of the invention does not contain acid as an additive, morepreferably does not contain acid.

In the method for the inhibition of glycolysis and optionallycoagulation in a blood sample in vitro blood is mixed after itswithdrawal with the composition according to the invention.

Another aspect of the present invention relates to an in vitrodiagnostic method, wherein the amount(s) of blood component(s) is (are)determined in a stabilized blood sample mixed with the compositionaccording to the present invention. In particular, one and/or more ofglucose, lactate and homocysteine are stabilized, preferably at roomtemperature for up to 50 hours after blood collection. Preferablyglucose, lactate and homocysteine are stabilized simultaneously. Thedetermination of the amount(s) of the respective blood component(s) iscarried out using conventional physical, chemical, enzymatic and/orimmunological methods, including combinations thereof.

A further aspect of the present invention is the use of the compositionof the invention for the inhibition of the glycolytic enzyme system andglycolysis and optionally coagulation in a blood sample in vitro.Unexpectedly, it was found in the present invention that the use of thecomposition of the present invention efficiently stabilizes any one ofglucose, lactate and homocysteine, and any combination thereof, inblood. Even more surprisingly, it was found in the present inventionthat the use of the composition of the present invention efficiently andadvantageously stabilizes glucose, lactate and homocysteine in bloodafter collection simultaneously. This allows blood glucose, lactate andhomocysteine to be determined accurately and reliably using a singlesample. According to this aspect of the invention, glucose, lactateand/or homocysteine can be preferably and advantageously stored andtransported at room temperature for up to 50 hours, more preferably forup to 72 hours, most preferably for 96 hours after blood collection.

By using antiglycolytic agents according to the present invention and inaddition anticoagulant, a blood sample can be stored and transported ina substantially physiologically native state. Room temperature denotes atemperature range from 20° C. to 25° C. However, a temperature above orbelow room temperature can also be acceptable, and a temperature rangefrom 0° C. to 37° C. lies within the use of the present invention.Moreover and preferably, concurrently with or subsequently to the use ofthe composition according to the present invention (a) test(s) is (are)carried out for the determination of one and/or more of glucose, lactateand homocysteine and optionally a further blood component.Advantageously a single sample can be used for a multi-analyte analysis,and possible interference from components of the composition of theinvention in the analytical testing for glucose, lactate andhomocysteine and other co-analytes is preferably substantially avoidedand more preferably avoided by choosing components that are compliant orrespectively non-interfering in the subsequent tests. The compositionaccording to the present invention can be advantageously used toincrease the reliability of diagnosis and prediction. In view of thepotential risk of hemolysis and the possible interference with theanalyses therefrom, according to a preferred embodiment the ammoniumsalt iv) is furthermore added to the composition.

Another aspect of the present invention is a blood collection devicecomprising the composition of the present invention, wherein the bloodcollection device preferably comprises a device which is capable ofbeing connected with a conventional blood withdrawal device.Conventional blood collection tubes including evacuated blood collectiontubes such as vacutainer and aspiration systems such as monovette areknown in the art.

In another aspect the invention relates to a use of the blood collectiondevice according to the present invention for stabilizing and/or storingof a blood sample in vitro, wherein preferably the blood sample isstabilized and/or stored at room temperature for up to 50 hours afterblood collection. However, a temperature range from 0° C. to 37° C. anda storage time of up to 96 hours are comprised in the invention. Thisaspect of the present invention provides a particularly efficient use,wherein concurrently or subsequently (a) test(s) is (are) preferablycarried out for the determination of one and/or more of glucose, lactateand homocysteine and optionally a further blood component.

A blood collection device comprising, placed in the device, acomposition according to the present invention is provided, followed byplacing blood in said blood collection device. Alternatively, blood isplaced in a blood collection device and subsequently a composition ofthe present invention is placed in the blood collection device. Thecomposition of the present invention is mixed with blood in the bloodcollection device. Optionally the blood is stored in the bloodcollection device for a predetermined period of time, for example untilprocessing such as centrifugation or analysis and for up to 50, 72 or 96hours at room temperature, during which glucose, lactate andhomocysteine levels are substantially constant. In a further step theamount of one and/or more of glucose, lactate and homocysteine and/orother blood component(s) in the blood sample is determined, for exampleby using conventional physical, chemical, enzymatic and/or immunologicalmethods, including combinations thereof. Without being limited thereto,analytical methods may comprise gas chromatography (GC), massspectrometry (MS), gas chromatography-mass spectrometry (GC-MS), gaschromatography-mass spectrometry with isotopic dilution (GC-ID-MS),liquid chromatography (LC), liquid chromatography with mass spectrometry(LC-MS), liquid chromatography with tandem mass spectrometry (LC-MS-MS),high-pressure liquid chromatography (HPLC), high-pressure liquidchromatography with fluorescence detection (HPLC-FD), HPLC withelectrochemical detection (HPLC-ED), fluorescence polarizationimmunoassay (FPIA), chemiluminescence immunoassay (e.g. ICL), enzymelinked immunoassay (EIA), ion-exchange chromatography (IEC), capillaryelectrophoresis and capillary electrophoresis with laser-inducedfluorescence, wherein HPLC and immunoassays are particularly widely usedin clinical laboratories.

After the mixing of the composition of the present invention and theblood, the mass concentration of the at least one inhibitor ofhexokinase preferably is at least 0.01 mg/mL blood, more preferably isat least 0.02 mg/mL blood, even more preferably is in a range from 0.02to 25 mg/mL blood and most preferably is in a range from 0.02 to 4 mg/mLblood, the mass concentration of the at least one glycolysis-inhibitingagent preferably is at least 0.01 mg/mL blood, more preferably is atleast 0.02 mg/mL blood, even more preferably is in a range from 0.02 to25 mg/mL blood and most preferably is in a range from 0.02 to 4 mg/mLblood, optionally the mass concentration of the optional anticoagulantpreferably is at least 0.01 mg/mL blood, more preferably is at least 0.1mg/mL blood, even more preferably is in a range from 0.1 to 25 mg/mLblood and most preferably is in a range from 1 to 2.5 mg/mL blood or 12to 30 IU/mL blood, and preferably the ratio of the mass concentrationsof the at least one inhibitor of hexokinase and the at least oneglycolysis-inhibiting agent is in a range from 100:1 to 1:100, morepreferably is in a range from 50:1 to 1:50, even more preferably is in arange from 25:1 to 1:25 and most preferably is in a range from 10:1 to1:10. Preferably, whole blood is mixed, and preferably plasma glucose,plasma lactate and plasma homocysteine respectively are determined.Using whole blood and plasma analysis instead of serum is advantageousin that there is no time required to wait for coagulation, there is moresample material and there are no artifacts stemming from coagulationeffects such as slight hemolysis or ongoing coagulation aftercentrifugation. Substantially constant levels preferably are levelswhich have a time-dependent variation of not more than 4% for glucoseand homocysteine and not more than 8% for lactate, more preferably arelevels which have a time-dependent variation of not more than 3% forglucose and homocysteine and not more than 5% for lactate, even morepreferably are levels which have a time-dependent variation of not morethan 2% for glucose and homocysteine and not more than 4% for lactate,yet even more preferably are levels which have a time-dependentvariation of not more than 1.5% for glucose and homocysteine and notmore than 3% for lactate and most preferably are levels which have atime-dependent variation of not more than 1% for glucose andhomocysteine and not more than 2% for lactate.

Another aspect of the present invention relates to a kit which comprisesthe blood collection device of the present invention and test substancesfor the determination of at least one of, optionally all simultaneously,glucose, lactate and homocysteine and optionally a further bloodcomponent in collected blood.

A further aspect of the invention is a composition as set forth in anyone of items (1) to (14) for the stabilization of one and/or more ofglucose, lactate and homocysteine in blood.

In an embodiment the at least one glycolysis-inhibiting agent ii) isNH₄F. The provision of NH₄F is particularly efficient because fluorideion can contribute to the inhibition of glycolysis, while advantageouslyat the same time ammonium ion can contribute to inhibiting hemolysis. Inparticular, fluoride salt is preferably ammonium fluoride becauseammonium fluoride can minimize changes in osmolarity and can avoid theoutflow of water from the blood cells. Thus a possible dilution effectand possible impairment of cellular integrity due to the desiccation ofblood cells can be inhibited.

In another aspect the present invention discloses a use of at least oneglycolysis-inhibiting agent for the stabilization and ex vivodetermination of homocysteine in blood after withdrawal, wherein the atleast one glycolysis-inhibiting agent preferably has activity for anenzyme involved in glucose catabolism, more preferably hasantiglycolytic activity for any one of the enzymes in the glycolyticpathway, and most preferably is at least one inhibitor of hexokinase.

Surprisingly, it was found in the present invention that the use of atleast one glycolysis-inhibiting agent, and in particular thecombinations of agents according to the present invention, not onlystabilizes glucose and lactate, but also homocysteine which is acomponent in sulfur amino acid metabolism. Unexpectedly, the sufficientand improved inhibition of glycolysis inhibits the metabolic processesthat lead to the ex vivo formation of homocysteine, presumably involvinga change in or an inhibition of intracellular ATP-dependent metabolicprocesses such as transmethylation reactions of homocysteine precursors.

In the present invention it is considered that particular agents canhave a multitude of functions and effects. For example, anantiglycolytic agent can inhibit and/or stimulate several enzymesinvolved in glucose catabolism at the same time, or also inhibithemolysis. Such bi- or multi-functionalities are understood to becomprised in the present invention. However, in typical and usuallyapplied embodiments of the present invention, each of these agents isspecific and monofunctional for the respective enzyme, i.e. at least onecompound for component i), at least another compound for component ii),and optionally again another compound for the optional component iii) ofthe composition as set forth in claim 1 are present, while furthermoreoptionally ammonium salt NR₄X iv) as set forth in claim 3 is present.

The following examples are merely illustrative of the present inventionand they should not be considered as limiting the scope of the inventionin any way. The examples and modifications or other equivalents thereofwill become apparent to those skilled in the art in the light of thepresent entire disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows glucose stability at room temperature in whole bloodcontaining A: 2-deoxy-D-glucose, ammonium fluoride and ammoniumheparinate; B: 2-deoxy-D-glucose, ammonium fluoride and sodiumheparinate; C: 2-deoxy-D-glucose, ammonium fluoride and potassium saltof EDTA; and D: potassium salt of EDTA.

FIG. 2 depicts lactate stability at room temperature in whole bloodcontaining A: 2-deoxy-D-glucose, ammonium fluoride and ammoniumheparinate; B: 2-deoxy-D-glucose, ammonium fluoride and sodiumheparinate; C: 2-deoxy-D-glucose, ammonium fluoride and potassium saltof EDTA; and D: potassium salt of EDTA.

FIG. 3 shows homocysteine stability at room temperature in whole bloodcontaining A: 2-deoxy-D-glucose, ammonium fluoride and ammoniumheparinate; B: 2-deoxy-D-glucose, ammonium fluoride and sodiumheparinate; C: 2-deoxy-D-glucose, ammonium fluoride and potassium saltof EDTA; and D: potassium salt of EDTA.

EXAMPLES AND COMPARATIVE EXAMPLES

Materials Used and Method

Materials

Iodoacetate, ammonium fluoride, dichloroacetic acid, 3-bromopyruvicacid, 2-deoxy-D-glucose, and sodium oxamate were purchased from SIGMA(SIGMA-ALDICH, Germany). Blood collection tubes with anticoagulants wereprovided by KABE (KABE Labortechnik GmbH, Germany)

Blood Collection and Sampling Protocol

Before blood collection, particular mixtures of antiglycolytic agentswere added into blood collection tubes containing anticoagulant. Therespective concentrations of the antiglycolytic agents in the mixtureswere in a range from 0.02 mg to 4 mg/mL of blood to be mixed with.Anticoagulant used was ammonium, lithium or sodium heparinate at aconcentration of 12.5 IU/mL or dipotassium ethylene diamine tetraaceticacid (K2EDTA) at a concentration of 2 mg/mL.

Blood was drawn from healthy volunteers by venipuncture of theantecubital vein by aspiration. Blood samples were collected into tubeswith or without the particular mixtures of antiglycolytic agents.Caution was taken that all tubes were filled to the mark and that theblood was well mixed with the agents by inverting the tubes 4 timesimmediately after blood collection.

After whole blood was mixed with different antiglycolytic agents and/oranticoagulants in the tubes, the tubes were stored at room temperature(from 20 to 25° C.) for different time intervals before centrifugation.The tubes were centrifuged 4, 15, 24, 48 and 50 hours after bloodcollection, and the separated plasma was stored at −20° C. untilanalysis. Concentrations of glucose, lactate and homocysteine weredetermined for the samples centrifuged after the different timeintervals of storage.

As a control and for comparison, a blood sample without anyantiglycolytic agent was centrifuged immediately after blood collectionand the plasma was separated within 10 min and stored at −20° C. untilanalysis. Glucose, lactate and homocysteine concentrations weredetermined for this sample, said concentrations being defined as thereference concentrations at time 0. Percentages for the time-dependentglucose, lactate and homocysteine concentrations relative to thereference concentrations (baseline) were computed.

Measurement of Glucose

The hexokinase method on cobas c (Roche Diagnostics) was used accordingto the protocol of the manufacturer.

Measurement of Lactate

A colorimetric method on cobas c (Roche Diagnostics) was used. Themethod is based on the oxidation of lactate to pyruvate by lactateoxidase.

Measurement of Total Homocysteine (tHcy)

tHcy was measured by a competitive immunoassay on IMMULITE (SIEMENS,Germany) and by the Diazyme Homocysteine Enzymatic Assay Kit on cobas c(Roche Diagnostics).

Example 1

Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood), ammoniumfluoride (2.9 mg/mL blood) and an anticoagulant in different aqueoussolutions (solvents were Solvent 1: H₂O; and Solvent 2: aq. ac.=weaklyacidified aqueous solution, respectively). After different timeintervals glucose, lactate and homocysteine concentrations weredetermined. The determined values are shown in Table 1 as percentages ofthe baseline. The combination of 2-deoxy-D-glucose and ammonium fluoridestabilized glucose, lactate and homocysteine fast, efficiently andcontinuously.

TABLE 1 changes in plasma analyte concentration time intervals (hours)analyte solvent 0 4 15 24 48 glucose H₂O 100 101 101 100 101 glucose aq.ac. 100 102 100 102 102 lactate H₂O 100 107 107 108 107 lactate aq. ac.100 102 104 104 104 homocysteine H₂O 100 100 100 102 100 homocysteineaq. ac. 100 101 101 100 100

For whole blood with 2-deoxy-D-glucose and ammonium fluoride, ammoniumheparinate (A), sodium heparinate (B) and potassium salt of EDTA (C)were used respectively as anticoagulant. For the anticoagulants A-C itwas found that glucose, lactate and homocysteine were stabilized fast,efficiently and continuously, as can be seen in FIGS. 1-3.

Example 2

Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood), sodiumiodoacetate (1.13 mg/mL blood) and an anticoagulant in different aqueoussolutions (solvents were Solvent 1: H₂O; and Solvent 2: aq. ac.=weaklyacidified aqueous solution, respectively). After different timeintervals glucose, lactate and homocysteine concentrations weredetermined. The determined values are shown in Table 2 as percentages ofthe baseline. The combination of 2-deoxy-D-glucose and sodiumiodoacetate stabilized glucose, lactate and homocysteine fast,efficiently and continuously.

TABLE 2 changes in plasma analyte concentration time intervals (hours)analyte solvent 0 4 15 24 48 glucose H₂O 100 100 100 101 100 glucose aq.ac. 100 101 102 101 101 lactate H₂O 100 97 97 98 97 lactate aq. ac. 100101 102 101 101 homocysteine H₂O 100 100 100 101 100 homocysteine aq.ac. 100 103 103 101 103

Example 3

Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood), sodiumoxamate (1.25 mg/mL blood) and an anticoagulant in weakly acidifiedaqueous solution as solvent. After different time intervals glucose andhomocysteine concentrations were determined. The determined values areshown in Table 3 as percentages of the baseline. The combination of2-deoxy-D-glucose and sodium oxamate stabilized glucose and homocysteinefast, efficiently and continuously.

TABLE 3 changes in plasma analyte concentration time intervals (hours)analyte 0 4 15 24 48 glucose 100 100 100 100 100 homocysteine 100 102108 112 112

Example 4

Whole blood was added to 3-bromopyruvic acid (2.5 mg/mL blood), sodiumiodoacetate (1.13 mg/mL blood) and an anticoagulant in water and weaklyacidified aqueous solution as solvent respectively. After different timeintervals glucose concentrations were determined. The determined valuesare shown in Table 4 as percentages of the baseline. The combination of3-bromopyruvic acid and sodium iodoacetate stabilized glucose fast,efficiently and continuously.

TABLE 4 changes in plasma glucose concentration time intervals (hours)solvent 0 4 15 24 48 H₂O 100 100 101 100 100 aq. ac. 100 104 104 104 104

Example 5

Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood), ammoniumfluoride (2.9 mg/mL blood), sodium oxamate (0.81 mg/mL blood) and ananticoagulant in weakly acidified aqueous solution as solvent. Afterdifferent time intervals glucose, lactate and homocysteineconcentrations were determined. The determined values are shown in Table5 as percentages of the baseline. The combination of 2-deoxy-D-glucose,ammonium fluoride and sodium oxamate stabilized glucose, lactate andhomocysteine fast, efficiently and continuously.

TABLE 5 changes in plasma analyte concentration time intervals (hours)analyte 0 4 15 24 48 glucose 100 102 102 103 102 lactate 100 96 96 96 97homocysteine 100 104 103 104 103

Example 6

Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood), sodiumiodoacetate (1.2 mg/mL blood), sodium oxamate (0.65 mg/mL blood) and ananticoagulant in weakly acidified aqueous solution as solvent. Afterdifferent time intervals glucose and lactate concentrations weredetermined. The determined values are shown in Table 6 as percentages ofthe baseline. The combination of 2-deoxy-D-glucose, sodium iodoacetateand sodium oxamate stabilized glucose and lactate fast, efficiently andcontinuously.

TABLE 6 changes in plasma analyte concentration time intervals (hours)analyte 0 4 15 24 48 glucose 100 102 101 102 102 lactate 100 98 97 98 98

Comparative Example 1

When whole blood was mixed with potassium salt of EDTA only, i.e. noantiglycolytic agent was added, glucose, lactate and homocysteine werenot stabilized. The glucose concentration decreased rapidly andcontinuously (see FIG. 1), while the lactate and homocysteineconcentrations increased rapidly and continuously (see FIGS. 2-3).

Comparative Example 2

Whole blood was added to Sarstedt blood collection tubes containingsodium fluoride and potassium oxalate. After different time intervalsglucose and lactate concentrations were determined. The determinedvalues are shown in Table 7 as percentages of the baseline. Glucose andlactate concentrations were not sufficiently stabilized, in any eventremarkably less than in the Examples according to the present invention.The glucose concentration decreased significantly and continuously,while the lactate concentration increased significantly andcontinuously.

TABLE 7 changes in plasma analyte concentration time intervals (hours)analyte 0 4 15 24 48 glucose 100 96 94 92 91 lactate 100 106 110 113 116

Comparative Example 3

Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood) and ananticoagulant in weakly acidified aqueous solution as solvent. Afterdifferent time intervals glucose and homocysteine concentrations weredetermined. The determined values are shown in Table 8 as percentages ofthe baseline. Glucose and homocysteine concentrations were notsufficiently stabilized. The glucose concentration decreasedsignificantly and continuously, while the homocysteine concentrationincreased significantly and continuously.

TABLE 8 changes in plasma analyte concentration time intervals (hours)analyte 0 4 15 24 48 glucose 100 87 85 80 80 homocysteine 100 102 106117 117

Comparative Example 4

Whole blood was added to 3-bromopyruvic acid (2.5 mg/mL blood) and ananticoagulant in weakly acidified aqueous solution as solvent. Afterdifferent time intervals glucose and homocysteine concentrations weredetermined. The determined values are shown in Table 9 as percentages ofthe baseline. Glucose and homocysteine concentrations were notsufficiently stabilized. The glucose concentration decreasedsignificantly and continuously, while the homocysteine concentrationincreased significantly and continuously.

TABLE 9 changes in plasma analyte concentration time intervals (hours)analyte 0 4 15 24 48 glucose 100 90 89 88.8 88 homocysteine 100 101 111111 111

A comparison of the Examples with the Comparative Examples shows thatthe compositions according to the present invention stabilize glucose,lactate and homocysteine fast, efficiently and continuously, whereascompositions not containing the particular combinations of agentsaccording to the present invention are insufficient in stabilizingglucose, lactate and homocysteine—even when agents are used that inhibithexokinase such as 2-deoxy-D-glucose (see Comparative Example 3) and3-bromopyruvic acid (see Comparative Example 4) alone.

Examples 7-13 and Comparative Example 5

In vitro hemolysis was tested in blood samples incubated at roomtemperature (22° C.-25° C.) by determining the free plasma hemoglobinconcentration (g/L) over time (0-50 hours), wherein blood samples weremixed with different compositions.

Example 7

Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, sodiumoxamate and K₃EDTA.

Example 8

Whole blood was added to 2-deoxy-D-glucose, ammonium salt of iodoaceticacid, sodium oxamate and K₃EDTA.

Example 9

Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, ammoniumsalt of oxamic acid and K₃EDTA.

Example 10

Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, sodiumoxamate, K₃EDTA and tetraethylammonium chloride.

Example 11

Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, sodiumoxamate, K₃EDTA and tetramethylammonium fluoride.

Example 12

Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, sodiumoxamate, K₃EDTA and tetramethylammonium chloride.

Example 13

Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, sodiumoxamate, K₃EDTA and ammonium chloride.

Comparative Example 5

Whole Blood was added to K₃EDTA

The concentrations of antiglycolytic agents in the mixtures were 0.1-4mg/mL blood. In the cases of Examples 10-13, 0.5-1 μmol of therespective ammonium salts per mL blood were added to the mixture.

TABLE 10 Free plasma hemoglobin concentration (g/L) time intervals(hours) 0 1.5 3.5 50 Comparative 0.290 0.290 0.300 0.370 Example 5Example 7 0.270 0.275 0.290 0.330 Example 8 0.200 0.210 0.210 0.210Example 9 0.245 0.250 0.250 0.250 Example 10 0.150 0.150 0.154 0.160Example 11 0.220 0.220 0.230 0.232 Example 12 0.280 0.280 0.280 0.280Example 13 0.290 0.290 0.290 0.290

Comparative Example 5 shows that when whole blood is mixed with onlyK₃EDTA, over time a significant degree of hemolysis occurs, as seen inthe increase of the free plasma hemoglobin concentration at 3.5 hoursand especially at 50 hours.

In Example 7, which does not contain any ammonium salt, hemolysis isalso observed, but to a lesser extent at 50 hours compared toComparative Example 5.

Examples 8-9 demonstrate that when the combination of antiglycolyticagents comprises salts, hemolysis can unexpectedly and advantageously besignificantly inhibited by providing at least one salt of said agents asan ammonium salt.

Surprisingly, hemolysis can also be efficiently and effectivelyinhibited by adding to the composition as a further component anammonium salt, as shown in Examples 10-13.

Therefore, the provision of an ammonium salt, as one or more of theantiglycolytic agents according to the invention and/or as a furtheradditive, provides significant further benefits. One and/or more ofglucose, lactate and homocysteine, preferably all, in the blood sampleis (are) stabilized, while furthermore the inhibition of hemolysis canbe significantly enhanced. This particularly advantageous stabilizationand preservation of the blood samples in turn can facilitate an improveddetermination of blood components and reliable diagnostics.

The invention claimed is:
 1. A blood collection device comprising acomposition comprising in combination and commonly placed in the bloodcollection device: i) at least one inhibitor of hexokinase, selectedfrom the group consisting of 2-deoxy-D-glucose,2-fluoro-2-deoxy-D-glucose, 2-amino-2-deoxy-D-glucose and 3-bromopyruvicacid or salt thereof; ii) at least one glycolysis-inhibiting agenthaving activity for another enzyme involved in glucose catabolism,selected from the group consisting of fluoride salt, iodoacetic acid orsalt therof, oxamic acid or salt thereof and dichloroacetic acid or salttherof; and optionally iii) an anticoagulant and/or a plasma stabilizer,selected from the group consisting of EDTA salt, citrate salt, oxalatesalt and heparin salt.
 2. The blood collection device according to claim1, wherein the composition further comprises iv) ammonium salt NR₄X,wherein each R independently is hydrogen, linear C₁-C₆ alkyl, branchedC₃-C₆ alkyl, unsubstituted phenyl or substituted phenyl, and X ishalide, hydroxide, C₁-C₄ alkoxide and acetate.
 3. The blood collectiondevice according to claim 1, wherein the at least oneglycolysis-inhibiting agent ii) has antiglycolytic activity for any oneof the enzymes in the glycolytic pathway downstream of hexokinase. 4.The blood collection device according to claim 1, further comprising ablood sample which is preserved for further analysis.
 5. The bloodcollection device according to claim 4, wherein one and/or more ofglucose, lactate and homocysteine in the blood sample is stabilized. 6.The blood collection device according to claim 4, wherein the massconcentration of the at least one inhibitor of hexokinase i) is at least0.01 mg/mL blood sample and wherein the mass concentration of the atleast one glycolysis-inhibiting agent ii) is at least 0.01 mg/mL bloodsample.
 7. The blood collection device according to claim 1, wherein thecomposition is substantially free of agents for the lysis of bloodcells.
 8. The blood collection device according to claim 1, comprising adevice which is capable of being connected with a conventional bloodwithdrawal device.
 9. The blood collection device according to claim 1,which is a blood collection tube, an evacuated blood collection tube, avacutainer or an aspiration system respectively enclosing saidcomposition.
 10. The blood collection device according to claim 1,combined within a kit further comprising test substances for thedetermination of at least one of, optionally all simultaneously,glucose, lactate and homocysteine and optionally a further bloodcomponent in collected blood.